Tropical and temperate wastewater treatment plants assemble different and diverse microbiomes.

The diversity and assembly of activated sludge microbiomes play a key role in the performances of municipal wastewater treatment plants (WWTPs), which are the most widely applied biotechnological process systems. In this study, we investigated the microbiomes of municipal WWTPs in Bangkok, Wuhan, and Beijing that respectively represent tropical, subtropical, and temperate climate regions, and also explored how microbiomes assembled in these municipal WWTPs. Our results showed that the microbiomes from these municipal WWTPs were significantly different. The assembly of microbiomes in municipal WWTPs followed deterministic and stochastic processes governed by geographical location, temperature, and nutrients. We found that both taxonomic and phylogenetic α-diversities of tropical Bangkok municipal WWTPs were the highest and were rich in yet-to-be-identified microbial taxa. Nitrospirae and ß-Proteobacteria were more abundant in tropical municipal WWTPs, but did not result in better removal efficiencies of ammonium and total nitrogen. Overall, these results suggest that tropical and temperate municipal WWTPs harbored diverse and unique microbial resources, and the municipal WWTP microbiomes were assembled with different processes. Implications of these findings for designing and running tropical municipal WWTPs were discussed. KEY POINTS: • Six WWTPs of tropical Thailand and subtropical and temperate China were investigated. • Tropical Bangkok WWTPs had more diverse and yet-to-be-identified microbial taxa. • Microbiome assembly processes were associated with geographical location.

Structural and metabolic adaptation of cellulolytic microcosm in co-digested Napier grass-swine manure and its application in enhancing thermophilic biogas production.

Biogas production from cellulosic wastes has received increasing attention. However, its efficiency is limited by the recalcitrant nature of plant cell wall materials. In this study, an active and structurally stable lignocellulolytic microcosm (PLMC) was isolated from seed culture in sugarcane bagasse compost by successive enrichment on Napier grass supplemented with swine manure, which is a mixture of highly fibrous co-digested waste under septic conditions. Tagged 16S rRNA gene sequencing on an Ion PGM platform revealed the adaptive merging of microorganisms in the co-digested substrates resulting in a stable symbiotic consortium comprising anaerobic cellulolytic clostridia stably co-existing with facultative (hemi)cellulolytic bacteria in the background of native microflora in the substrates. Ethanoligenens, Tepidimicrobium, Clostridium, Coprococcus, and Ruminococcus were the most predominant taxonomic groups comprising 72.82% of the total community. The remarkable enrichment of catabolic genes encoding for endo-cellulases and hemicellulases, both of which are key accessory enzymes in PLMC, was predicted by PICRUSt. PLMC was capable of degrading 43.6% g VS and 36.8% g VSS of the co-digested substrates within 7 days at 55 °C. Inoculation of the microcosm to batch thermophilic anaerobic digestion containing both substrates led to a 36.6% increase in methane yield along with an increase in cellulose removal efficiency. This study demonstrated structural and metabolic adaptation of the cellulolytic microcosms isolated in the background of native microflora from the co-digested wastes and its potent application in the enhancement of anaerobic digestion efficiency.

Characterization of cellulolytic microbial consortium enriched on Napier grass using metagenomic approaches.

Energy grass is a promising substrate for production of biogas by anaerobic digestion. However, the conversion efficiency is limited by the enzymatically recalcitrant nature of cellulosic wastes. In this study, an active, structurally stable mesophilic lignocellulolytic degrading microbial consortium (Np-LMC) was constructed from forest compost soil microbiota by successive subcultivation on Napier grass under facultative anoxic conditions. According to tagged 16S rRNA gene amplicon sequencing, increasing abundance of facultative Proteobacteria was found in the middle of batch cycle which was then subsequently replaced by the cellulose degraders Firmicutes and Bacteroidetes along with decreasing CMCase, xylanase, and ß-glucanase activity profiles in the supernatant after 5 days of incubation. Anaerobic/facultative bacteria Dysgonomonas and Sedimentibacter and aerobic bacteria Comamonas were the major genera found in Np-LMC. The consortium was active on degradation of the native and delignified grass. Direct shotgun sequencing of the consortium metagenome revealed relatively high abundance of genes encoding for various lignocellulose degrading enzymes in 23 glycosyl hydrolase (GH) families compared to previously reported cellulolytic microbial communities in mammalian digestive tracts. Enzymes attacking cellulose and hemicellulose were dominated by GH2, 3, 5, 9, 10, 26, 28 and 43 in addition to a variety of carbohydrate esterases (CE) and auxiliary activities (AA), reflecting adaptation of the enzyme systems to the native herbaceous substrate. The consortium identified here represents the microcosm specifically bred on energy grass, with potential for enhancing degradation of fibrous substrates in bioenergy industry.

Effect of freeze-thaw process on physical properties, microbial activities and population structures of anaerobic sludge.

Effects of the freeze-thaw process on physical properties, cell viability, microbial activities and population structures of anaerobic sludge were investigated. It was found that the sludge volume index was greatly reduced from 16.4 mL/g in the original sludge to 4.0 mL/g in the solid fraction of the frozen-thawed sludge. Even though the freeze-thaw process decreased cell viability in the solid fraction of the frozen-thawed sludge, microbial activity tests showed that the freeze-thaw process enhanced acidogenic activity approximately 20%. The enhanced acidogenic activity of the solid fraction was in good agreement with the enrichment of Clostridiaceae, Porphyromonadaceae and Propionibacteriaceae found in the solid fraction. The relative abundances of Proteobacteria families Oxalobacteraceae, Moraxellaceae, and Pseudomonadaceae were found to be highest in the liquid fraction where they form a substantial proportion of the bacterial community (a total of 59%).

Survey of Microbial Diversity in Flood Areas during Thailand 2011 Flood Crisis Using High-Throughput Tagged Amplicon Pyrosequencing.

The Thailand flood crisis in 2011 was one of the largest recorded floods in modern history, causing enormous damage to the economy and ecological habitats of the country. In this study, bacterial and fungal diversity in sediments and waters collected from ten flood areas in Bangkok and its suburbs, covering residential and agricultural areas, were analyzed using high-throughput 454 pyrosequencing of 16S rRNA gene and internal transcribed spacer sequences. Analysis of microbial community showed differences in taxa distribution in water and sediment with variations in the diversity of saprophytic microbes and sulfate/nitrate reducers among sampling locations, suggesting differences in microbial activity in the habitats. Overall, Proteobacteria represented a major bacterial group in waters, while this group co-existed with Firmicutes, Bacteroidetes, and Actinobacteria in sediments. Anaeromyxobacter, Steroidobacter, and Geobacter were the dominant bacterial genera in sediments, while Sulfuricurvum, Thiovirga, and Hydrogenophaga predominated in waters. For fungi in sediments, Ascomycota, Glomeromycota, and Basidiomycota, particularly in genera Philipsia, Rozella, and Acaulospora, were most frequently detected. Chytridiomycota and Ascomycota were the major fungal phyla, and Rhizophlyctis and Mortierella were the most frequently detected fungal genera in water. Diversity of sulfate-reducing bacteria, related to odor problems, was further investigated using analysis of the dsrB gene which indicated the presence of sulfate-reducing bacteria of families Desulfobacteraceae, Desulfobulbaceae, Syntrobacteraceae, and Desulfoarculaceae in the flood sediments. The work provides an insight into the diversity and function of microbes related to biological processes in flood areas.

Enhancement of biogas production from swine manure by a lignocellulolytic microbial consortium.

Anaerobic digestion of lignocellulosic wastes is limited by inefficient hydrolysis of recalcitrant substrates, leading to low biogas yield. In this study, the potential of a lignocellulolytic microbial consortium (LMC) for enhancing biogas production from fibre-rich swine manure (SM) was assessed. Biochemical methane potential assay showed that inoculation of structurally stable LMC to anaerobic digestion led to increase biogas production under mesophilic and thermophilic conditions. The greatest enhancement was observed at 37°C with a LMC/SM ratio of 1.5:1 mg VSS/g VS leading to biogas and methane yields of 355 and 180 ml/g VS(added) respectively, equivalent to 40% and 55% increases compared with the control. The LMC was shown to increase the efficiency of total solid, chemical oxygen demand removal and degradation of cellulose and hemicelluloses (1.87 and 1.65-fold, respectively). The LMC-supplemented process was stable over a 90 d biogas production period. This work demonstrates the potential of LMC for enhancing biogas from lignocellulosic wastes.

Comparative metagenomic analysis of microcosm structures and lignocellulolytic enzyme systems of symbiotic biomass-degrading consortia.

Decomposition of lignocelluloses by cooperative microbial actions is an essential process of carbon cycling in nature and provides a basis for biomass conversion to fuels and chemicals in biorefineries. In this study, structurally stable symbiotic aero-tolerant lignocellulose-degrading microbial consortia were obtained from biodiversified microflora present in industrial sugarcane bagasse pile (BGC-1), cow rumen fluid (CRC-1), and pulp mill activated sludge (ASC-1) by successive subcultivation on rice straw under facultative anoxic conditions. Tagged 16S rRNA gene pyrosequencing revealed that all isolated consortia originated from highly diverse environmental microflora shared similar composite phylum profiles comprising mainly Firmicutes, reflecting convergent adaptation of microcosm structures, however, with substantial differences at refined genus level. BGC-1 comprising cellulolytic Clostridium and Acetanaerobacterium in stable coexistence with ligninolytic Ureibacillus showed the highest capability on degradation of agricultural residues and industrial pulp waste with CMCase, xylanase, and ß-glucanase activities in the supernatant. Shotgun pyrosequencing of the BGC-1 metagenome indicated a markedly high relative abundance of genes encoding for glycosyl hydrolases, particularly for lignocellulytic enzymes in 26 families. The enzyme system comprised a unique composition of main-chain degrading and side-chain processing hydrolases, dominated by GH2, 3, 5, 9, 10, and 43, reflecting adaptation of enzyme profiles to the specific substrate. Gene mapping showed metabolic potential of BGC-1 for conversion of biomass sugars to various fermentation products of industrial importance. The symbiotic consortium is a promising simplified model for study of multispecies mechanisms on consolidated bioprocessing and a platform for discovering efficient synergistic enzyme systems for biotechnological application.

Alkaliphilic endoxylanase from lignocellulolytic microbial consortium metagenome for biobleaching of eucalyptus pulp.

Enzymatic pre-bleaching by modification of pulp fibers with xylanases is an attractive approach to reduce the consumption of toxic bleaching chemicals in the paper industry. In this study, an alkaliphilic endoxylanase gene was isolated from metagenomic DNA of a structurally stable thermophilic lignocellulose-degrading microbial consortium using amplification with conserved glycosyl hydrolase family 10 primers and subsequent genome walking. The full-length xylanase showed 78% sequence identity to an endo-beta-1,4-xylanase of Clostridium phytofermentans and was expressed in a mature form with an N-terminal His6 tag fusion in Escherichia coli. The recombinant xylanase Xyn3F was thermotolerant and alkaliphilic, working optimally at 65-70 degrees C with an optimal pH at 9- 10 and retaining >80% activity at pH 9, 60 degrees C for 1 h. Xyn3F showed a Vmax of 2,327 IU/mg and Km of 3.5 mg/ml on birchwood xylan. Pre-bleaching of industrial eucalyptus pulp with no prior pH adjustment (pH 9) using Xyn3F at 50 IU/g dried pulp led to 4.5-5.1% increase in final pulp brightness and 90.4-102.4% increase in whiteness after a single-step hypochlorite bleaching over the untreated pulp, which allowed at least 20% decrease in hypochlorite consumption to achieve the same final bleaching indices. The alkaliphilic xylanase is promising for application in an environmentally friendly bleaching step of kraft and soda pulps with no requirement for pH adjustment, leading to improved economic feasibility of the process.